We will prepare a replication map of a circular derivative of yeast chromosome III. By virtue of its structure the circular chromosome III is unique among chromosomal DNA's. The replication map will include information on the positions of initiation sites and their temporal sequence of activation and will allow us to determine whether initiation sites are specific and whether the temporal sequence of replication is constant. We will also identify cloned fragments of chromosome III capable of autonomous replication using the yeart transformation system and determine whether replication origins used in chromosome replication correspond to autonomously replicating sequences (ars). The cell cycle mutant cdc2 is able to replicate only about 80% of its genome at the nonpermissive temperature. We will determine whether cdc2 is defective in the replication of a specific fraction of its genome by density labelling DNA synthesized at 36 degrees and hybridizing chromosome-specific probes separately to the hybrid and light density peaks. We are isolating temperature sensitive mutants defective in mitochondrial DNA replication. The mutants will be used to determine how many genes are required for mtDNA replication and for isolating mtDNA replication intermediates for an electron microscopic analysis of their structure and position.